Lymphangioleiomyomatosis (LAM) is a genetic disorder characterized by widespread, potentially metastatic lesions of smooth muscle-like LAM cells promoting cystic destruction of the lung for which there is no therapy. Our previous studies in elucidating the molecular mechanism of LAM led to the identification of essential function of the tumor suppressor tuberous sclerosis complex 2 (TSC2) as a negative regulator of mammalian target of rapamycin (mTOR)/p70 S6 kinase (S6K1) in LAM. These pre-clinical findings led to a Phase 2 rapamycin clinical trial for LAM patients. In this project we propose to further elucidate the downstream components deregulated by the TSC2 loss that contribute to LAM and to explore a new therapeutic target for combination therapy with rapamycin in LAM. Current evidence suggests that LAM cells spread metastatically to distinct organs, a process requiring the coordinated functions of Rho GTPases and cell adhesion to promote migration and invasiveness. The precise mechanism regulating LAM cell migration remains unknown. Our preliminary data demonstrate that loss of TSC2 results in TSC1-dependent activation of RhoA and inhibition of Rac1, the upregulation of integrins, and increased cell migration and invasiveness, which are abolished by TSC2 re-expression or the inhibition of RhoA activity. Our preliminary data also show that RhoA activity contributes to increased LAM cell growth, and simvastatin, which inhibits RhoA activity, has synergistic, anti-proliferative and anti-tumor activities when combined with rapamycin. Based on this evidence, we hypothesize that TSC2 regulates actin cytoskeleton and cell adhesion via TSC1-mediated regulation of Rac1 and RhoA GTPases. We further propose that TSC2 suppresses cell migration and that loss of TSC2 in LAM cells promotes cell migration and invasiveness that contributes to LAM tumor growth. In Aim 1, we will determine the mechanism by which disease-associated TSC2 mutants dysregulate the activity of RhoA and Rac1 GTPases. In Aim 2, we will determine whether TSC2 and Rho GTPases modulate integrin expression, cell adhesion, migration and invasiveness, and identify the effector pathway(s) mediating these effects. Based on evidence that RhoA regulates cell motility and cell growth, in Aim 3, we will test our hypotheses that the combined inhibition of RhoA with simvastatin and rapamycin will abrogate estrogen-stimulated growth of TSC2-null tumors compared to the effects of each agent alone by using a xenographic model of rat TSC2-null cells in athymic nude mouse. Collectively, these studies will define the key cellular and molecular mechanisms by which TSC2 and Rho GTPase regulate LAM cell adhesion, migration, and invasiveness;moreover, our studies will provide insight into the combinational therapeutic targets that may prevent or abrogate tumor growth in LAM. PROJECT NARRATIVE: Lymphangioleiomyomatosis (LAM) is a deadly lung disease that targets only women, striking them down during their childbearing years and can be triggered by pregnancy, progresses rapidly, and often results in death within ten years. The disease causes extensive, abnormal smooth muscle-like cell proliferation, which invades and destroys the tissues of the lung by forming cysts, eventually obstructing the flow of air and leading to lung collapse and failure. This study will elucidate the role of tumor suppressor tuberous sclerosis complex 2 (TSC2) and Rho family of small GTPases in LAM and will identify novel targets for combinational pharmaceutical intervention to treat this disease.